Hsp104 is a protein chaperone that protects cells from stress by resolubilizing proteins from aggregates. This disaggregation activity is required for propagation of amyloid-based yeast prions. When Hsp104 expression is elevated under conditions where it is not normally induced it causes prions to be lost from cells. For over a decade a universally held view of the underlying process is that Hsp104 protein disaggregation activity dissolves prion aggregates until templates for further prion propagation are eliminated. We identified Hsp104 mutants that enhance prion propagation in both wild type and Hsp70 mutant cells. In characterizing these mutants we discovered that Hsp104's amino-terminal region (NTD), which is conserved but whose function is unknown, is required for prion elimination by overproduced Hsp104 but not for Hsp104 activities required for thermotolerance and prion propagation. Excess NTD alone does not cure prions. These findings imply that Hsp104's known functions are insufficient for eliminating prions. Thus, the mechanism of prion curing is not complete solubilization of prion aggregates by Hsp104 and an unknown Hsp104 function is required to cure prions. We are combining gene knockout and protein interaction screens to identify candidate factors that influence the ability of overproduced Hsp104 to eliminate prions. We have identified mutations in several genes that strongly or completely inhibit curing by overexpressed Hsp104, and we are characterizing their effects genetically to determine how they are involved in prion curing by Hsp104.[unreadable] For in vitro studies we are purifying wild type and mutant Hsp104 proteins to determine what enzymatic functions are affected by the Hsp104 mutations and how these altered activities affect Hsp104 chaperone machinery function in protein disaggregation. We will eventually determine how the mutations affect physical and functional interactions of Hsp104 with amyloid, alone or in combination with other chaperones. This work will help us understand how specific alterations of Hsp104 function influence prion propagation in vivo.